Electrospray ionization (ESI) converts involatile solutes into gas phase ions for mass spectrometry (MS), as introduced in U.S. Pat. Nos. 4,531,056; 5,581,080; 5,686,726; 6,118,120, among others. ESI ions may also be analyzed via alternative gas phase methods such as ion mobility spectrometry (IMS). ESI's tendency to form multiply charged ions is advantageous to widen the MS's mass range. However, as first noticed by S. F. Wong, C. K. Meng, and J. B. Fenn, (J. Phys. Chem. 1988, 92, 546-550), multiple charging greatly increases the number of peaks present, often resulting in unresolvable spectral complexity. For this reason, various charge-reduction techniques have been described, for instance by Reid G. E., Wells, J. M., Badman, E. R., McLuckey, S. A. (Int. J. Mass Spectrom. 2003, 222, 243-258), and by Scalf, M., Westphall, M. S., Krause, J., Kaufman, S. L. Smith, L. M., (Science, 1999, 283, 194-197). Maximal spectral simplification is evidently achieved by producing dominantly singly charged ions, as described in U.S. Pat. Nos. 5,076,097; 5,247,842; 6,544,484; 7,796,727. This drastic level of charge reduction is not easily made incompatible with the limited mass range of MS detectors in studies of large protein complexes and viruses. However, IMS permits the analysis of singly charged ions of considerable sizes: Certainly up to 30 nm with resolving powers approaching (and possibly exceeding) 40, as discussed by J. Fernandez de la Mora and J. Kozlowski (J. Aerosol Sci., 57, 45-53, 2013). Even larger sizes can be analyzed with some resolution concessions as in the work of Kaufman (J. Aerosol Sci. (29), 537-552, 1998). Furthermore, such massive ions can be detected individually at ambient conditions by growing them into visible drops via vapor condensation in so-called condensation nucleus counters CNCs (also called condensation particle counters, CPCs), as described in U.S. Pat. No. 4,790,650. This sensitive detector is not easily coupled to conventional drift time IMS systems due to its relatively slow response time (˜1 s). However, new IMS designs with response times >1 s have already shown promise in the protein size range (i.e. D. R. Oberreit, P. H. McMurry & C. J. Hogan Jr., Aerosol Sci. & Techn., 2014, 48:1, 108-118). When combined with faster CNCs (such as that of J. Wang, V. F. McNeill, D. R. Collins, R. C. Flagan, Aerosol Sci. & Techn., 2002, 36(6), 678-689) these fast IMS systems may improve their current resolving power approaching 20. Differential mobility analyzers (DMAs) following the design of Knutson and Whitby (J. Aerosol Sci., 1975, 6, 443-451) have long offered an alternative to IMS for mobility separation, and are in addition readily compatible with slow detectors. Therefore, the combination of a DMA with charge-reduced electrospray and a CNC detector has demonstrated considerable advantages for the analysis of large biological ions much harder to study by mass spectrometry. This combination, introduced in U.S. Pat. No. 5,076,097, has been termed GEMMA, and has been widely used in numerous applications, as discussed for instance by Guha et al. (Trends in Biotechnology, 2012, 30, 291-300); Kaddis, et al. (J. American Soc. Mass Spectrom., 2007, 18, 1206-1216); Bacher et al. (Mass Spectrom, 2001, 36, 1038-1052), or Maisser et al. (Phys. Chem. Chem. Phys., 2011, 13, 21630-21641), which will be further discussed and referred to as Maisser. The main shortcoming of GEMMA is its limited mobility resolution, typically with relative full width at half maximum (FWHM) >20% for proteins (i.e., FIG. 4a of S. L. Kaufman, J. Aerosol Sci. (29), 537-552, 1998). In contrast IMS spectra of electrosprayed proteins give relative full width at half maximum (FWHM) ˜3-4%, both in conventional drift time IMS and in studies based on high resolution DMAs, including cases with charge reduction (FIG. 2 of J. Fernandez de la Mora, S. Ude, B. A. Thomson, Biotechnol. J. 2006, 1, 988-997). This fact, together with the ability of DMAs to reach resolving powers as high as 100 (as in P. Martinez-Lozano and J. Fernandez de la Mora, J. Aerosol Sci., 2006, 37, 500-512), suggests that an improved GEMMA-like method should enable good ion transmission and a resolving power˜30 (FWHM˜3.33%), limited only by the natural coexistence of several gas phase protein conformations. Several authors [Fernandez de la Mora et al. Biotechnol. J. 2006, 1, 988-997; B. K. Ku, J. Fernandez de la Mora, D. A. Saucy, J. N. Alexander, Anal. Chem. 2004, 76, 814-822.; D. A. Saucy, S. Ude, I. W. Lenggoro, J. Fernandez de la Mora, Anal. Chem., 2004, 76, 1045-1053] have previously reasoned that the limited resolution of GEMMA results from its unconventional electrospraying method (connected to the charge reduction step), giving rise to a substantial and variable level of clustering of involatile residue material on the protein ion. However, the alternative ES-charge-reduction approaches they have proposed have not been widely adopted, perhaps because their charge reduction efficiency, transmission and peak width have not been sufficiently optimized or documented.
Recent signs of increased interest in higher resolution variants of GEMMA must be noted. Laschober, C. S. Kaddis, G. P. Reischl, J. A. Loo, G. Allmaier, W. W. Szymanski (J. Exp. Nanosci. 2007, 2(4) 291-301) have studied combinations of GEMMA's charge reduction with several DMAs and found clearly more compact protein structures for the DMA having a higher resolving power. More recently Maisser used another DMA of even higher resolving power, and found also modest peak width reductions with some native proteins: FWHM>17.6% for all but one (14.7% for ovalbumin). However, these authors discovered a considerable peak narrowing for several small and strongly denatured proteins, with FWHM as low as 8.5%. Good DMA resolution is hence necessary, but not sufficient to achieve narrow peaks.
The acidification advantage just discussed is of great intrinsic interest, offering superficial analogies with the well-known (but not so well understood) role of acids in reducing clustering in ESI-MS (and ESI-IMS). As suggested by Konermann, L., Ahadi, E., Rodriguez, A. D., Vahidi, S., (Anal. Chem., 2013, 85, 2-9), and by Consta, S., Malevanets, A. (Phys. Rev. Lett., 2012, 109, 148301) linear chains (including denatured proteins) can apparently be extruded cleanly out of a charged drop. In contrast, as noted in Fernandez de la Mora, J., Analytica Chimica Acta, 2000, 406, 93-104, globular proteins remain imprisoned in electrosprayed drops until the drops dry, so that the protein inherits the full load of involatile material originally carried by the drop. Unfortunately, the protein extrusion mechanisms of Consta or Konermann are unlikely to happen in singly or doubly charged drops, so the peak narrowing observed by Maisser calls for a different explanation. Acidification is in any case not a general antidote against an imperfect electrospray, first because it is not helpful in the case of protein complexes falling apart at unnatural pH, and also because the observed beneficial effect is minimal at protein masses beyond 40 kDa (FWHM=14.7%→13.4% for ovalbumin in Maisser). Another notable exception to GEMMA's generally wide peaks has been recently reported for viruses by R. You, M. Li, S. Guha, G. W. Mulholland, M. R. Zachariah, Anal Chem, 2014, 86, 6836-6842, who find FWHM in some cases below 5%. This exceptional narrowness perhaps follows from a cleaner virus preparation as well as the closer match between the diameters of the virus and the initial ES drop, which results in a relatively small level of adduction. In conclusion, it appears that much of the resolution problem noted in the case of proteins results from the unusual ESI conditions used in GEMMA. The ES-charge reduction process will therefore be the focus of the present invention.
The key to minimize spectral complexity is to reduce the charge state to unity (z=1), perhaps tolerating a small contribution of doubly charged ions (z=2). Here we shall start the discussion with charge-reduction methods involving the interaction of the ES ions with a bipolar mixture of singly charged anions and cations, produced by ionizing radiation (radioactive sources, UV, X rays) in an initially neutral gas at near ambient pressure. Because these sources produce dominantly monovalent ions, for multiply charged cations, the initial z evolves by interacting with monovalent anions, going sequentially through all the lower charge states z→z−1→z−2, . . . , →1. If insufficient reaction time is given, undesirable multiply charged ions survive. For an excessive reaction time, even singly charged ions are neutralized, leading to poor conversion into the z=1 product sought. An optimal reaction time t* may therefore be chosen to maximize the magnitude of the z=1 peak such that the probability of surviving z=2 ions is below a desired threshold, as described by J. Fernandez de la Mora, S. Ude, B. A. Thomson, in Biotechnol. J. 2006, 1, 988-997. As shown in that study, this optimal time depends weakly on the initial charge states zin as t*(zin)˜ln(zin), biasing slightly the signal intensity, and complicating quantification in complex mixtures. This difficulty has been ingeniously circumvented in the GEMMA design by tuning t* for the initial ES drops (before they undergo a first Coulomb explosion), which, as shown by de Juan, L., and J. Fernandez de la Mora, J. Coll. and Interface Sci, 1997, 186, 280-293, may be produced with relatively good uniformity of size and charge. The drawback of this early neutralization is that the volume of involatile residue that adducts to the final protein ions is that contained in the volume of the original ES drop, rather than that in the much smaller final drops produced by the usual long series of Coulomb explosions. This increased adduction decreases artificially the mobility, and widens the mobility peak in a fashion reflecting the width of the size distribution of the original ES drops. Accordingly, early neutralization is not ideal for resolution.
Early neutralization is not optimal either for sensitivity. Indeed, ES drops may be initially 10 nm in diameter, and may complete evaporation in sub-microsecond times. Accordingly, achieving drop neutralization prior to the first Coulomb explosion requires special measures, such as the relatively high humidity in the ES chamber recommended in U.S. Pat. No. 5,247,842, and the initial drops larger than those achievable in ES practice. For instance, Kaufman and colleagues (Anal. Chem. 1996, 68, 1895-1904; J. Aerosol Sci. 29, 537-552, 1998) use 20 mM aqueous ammonium acetate, while 100 mM (manageable in practice) would produce typical initial drop volumes 5 times smaller, as explained by Fernandez de la Mora in the Ann. Rev. Fluid Mechanics, 2007, 39, 217-243. The larger initial drop diameter and humidity used to delay drop evaporation also delay the production of analyte ions, resulting in higher space charge broadening and dilution of the ion cloud. Furthermore, the solution concentration must be tuned such that each final drop contains at most one analyte ion, forcing much smaller solution concentrations in initially large non-exploding drops than with initially small drops further atomized by Coulombic explosions. Therefore, both from the sensitivity and the resolution point of view, it is far better to produce the smallest possible ES drops, and evaporate them as completely and as swiftly as possible, as amply confirmed by the well known experience of so-called nanospray. Space charge dilution of the ion cloud evidently continues after complete drop drying, whence fast sampling into an analytical instrument is usually desirable. In our case the ions must first be charge-reduced, which decreases drastically the space charge field E as well as the analyte ion mobility Z (hence the space charge dilution velocity ZE). Accordingly, one should inject the analyte ions into the charge-reduction chamber immediately following complete drop drying, but not before. There is however a difficulty. The formation of a Taylor cone takes place ordinarily at the interface between a conducting fluid (the solution) and an insulating medium (the surrounding gas). If the electric field from the capillary tip penetrates into the charge-reduction region, some of the free ions present there are drawn into the electrospraying chamber, and the medium surrounding the Taylor cone ceases to be strictly insulating. Little is known on the physics of Taylor cone formation under such conditions, other than the readily observable fact that the range of stability of the electrospray is severely curtailed, very much as in situations where an electrical discharge forms at the liquid tip.
Note finally that analyte quantification (relating the measured gas phase concentration to the original solution concentration) when drying before neutralizing is in principle as viable as when neutralizing before drying. Both require corrections due to the size dependence of transport loses and charge-reduction efficiency (both losses are also charge-dependent, but only size counts since the charge on large biomolecules scales with the Rayleigh limit, approximately with the ½ power of molecular volume).